The motor neuron disease spinal muscular atrophy (SMA) is the leading inherited cause of death in infancy and childhood. It is caused by recessive mutations of the survival motor neuron 1 gene (SMN1), but all patients retain one or more copies of the homologous SMN2 gene that produce inadequate levels of SMN protein due to an alternative splice event. Novel therapeutics including splice-switching oligonucleotides (SSOs) can modulate this splice event thus increasing SMN expression. SSOs are currently being tested in phase III clinical trials in SMA patients, but they have a ceiling effect imposed by existing levels of SMN2 pre-mRNA. The efficacy of SMA splice-modulating treatments could be substantially accentuated by combining such treatments with specific SMN2 promoter activation. In preliminary data, we have identified a novel SMN2- associated long non-coding RNA (SMN-NAT), which is enriched in the CNS and represses SMN2 gene expression via recruitment of the epigenetic modifier Polycomb repressive complex 2 (PRC2). We have further shown that SMN-NAT-targeting antisense oligonucleotides (ASOs) can suppress SMN-NAT resulting in enhanced expression of SMN in cultured cells. In order to further explore whether SMN-NAT regulates SMN2 in human SMA patient cells and in neurons, in Specific Aim 1 we will examine the effects of SMN-NAT ASOs in several fibroblast cell lines derived from SMA patients and controls, in primary neurons derived from severe SMA mice, and in motor neurons derived from SMA induced pluripotent stem cells (iPSCs). In Specific Aim 2, we will further define the role of the PRC2 complex in the epigenetic control of SMN2 gene expression in SMA patient cells and neurons. Finally, in Specific Aim 3, we will examine the therapeutic potential of targeting SMN-NAT by treating severe SMA mice with SMN-NAT ASOs alone or in combination with SSOs and examining effects on SMN expression, behavioral outcomes, and motor unit histology. Together, these studies will characterize novel mechanisms of SMN2 gene control in neurons and determine whether targeting a SMN2-associated lncRNA could represent a new therapeutic strategy for SMA, which has the promise of working additively with splice modulating treatments.